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Heidi Häikiö for her contributions to this study. References. 1. Neel BG ... Forsell PA, Boie Y, Montalibet J, Collins S, Kennedy BP. Genomic characterization of ...
Protein Tyrosine Phosphatase 1B Variant Associated with Fat Distribution and Insulin Metabolism Olavi Ukkola,*† Tuomo Rankinen,* Timo Lakka,*†† Arthur S. Leon,‡ James S. Skinner,§ Jack H. Wilmore,¶ D.C. Rao,** Y.A. Kesa¨niemi,† and Claude Bouchard*

Abstract UKKOLA, OLAVI, TUOMO RANKINEN, TIMO LAKKA, ARTHUR S. LEON, JAMES S. SKINNER, ¨ NIEMI, JACK H. WILMORE, D.C. RAO, Y.A. KESA AND CLAUDE BOUCHARD. Protein tyrosine phosphatase 1B variant associated with fat distribution and insulin metabolism. Obes Res. 2005;13:829 – 834. Protein tyrosine phosphatase 1B (PTPN1) affects the regulation of insulin signaling and energy metabolism. We studied whether polymorphisms in the PTPN1 gene impact body fat distribution in the HERITAGE Family Study cohort in 502 white and 276 black subjects. Insulin sensitivity index, glucose disappearance index, acute insulin response to glucose (AIRglucose), and the disposition index (DI) were obtained from the frequently sampled intravenous glucose tolerance test. White subjects with the G82G at the PTPN1 IVS6⫹G82A polymorphism had higher body fat levels (p ⫽ 0.031) and sum of eight skinfolds (p ⫽ 0.003) and highest subcutaneous fat on the limbs (p ⫽ 0.002). G82A subjects had the lowest AIRglucose (p ⫽ 0.005) and disposition index (p ⫽ 0.040). Interaction effects between PTPN1 and leptin receptor gene variants influenced insulin sensitivity index and AIRglucose (p from 0.006 to 0.010). The

Received for review September 8, 2003. Accepted in final form March 8, 2005. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *Pennington Biomedical Research Center, Baton Rouge, Louisiana; †Department of Internal Medicine and Biocenter, University of Oulu, Oulu, Finland; ‡School of Kinesiology, University of Minnesota, Minneapolis, Minnesota; §Department of Kinesiology, Indiana University, Bloomington, Indiana; ¶Department of Kinesiology and Health Education, University of Texas at Austin, Austin, Texas; **Division of Biostatistics, Departments of Genetics and Psychiatry, Washington University School of Medicine, St. Louis, Missouri; and ††Kuopio Research Institute of Exercise Medicine, and Department of Physiology, University of Kuopio, Kuopio, Finland. Address correspondence to Claude Bouchard, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808-4124. E-mail: [email protected] Copyright © 2005 NAASO

variant PTPN1 Pro387Leu was associated with lower fasting insulin level (p ⫽ 0.035) and glucose disappearance index (p ⫽ 0.038). In summary, PTPN1 IVS6⫹G82G homozygotes showed higher levels of all measures of adiposity. G82 allele heterozygotes are potentially at higher risk for type 2 diabetes. Gene-gene interactions between the PTPN1 and leptin receptor genes contributed to the phenotypic variability of insulin sensitivity. The PTPN1 Pro387Leu variant was associated with lower glucose tolerance. Key words: genotype, adiposity, insulin sensitivity, protein tyrosine phosphate 1B

Protein tyrosine phosphatase 1B (PTPN1)1 represents the nontransmembrane form of the enzyme (1). Although overexpression of PTPN1 inhibits insulin signaling (2), inhibition of PTPN1 can enhance it (3). PTPN1 knockout mice show increased insulin action and resistance to obesity and diabetes (4). This unexpected combination has been recently explained by PTPN1’s negative influence on leptin signaling (5). Increased PTPN1 activity in skeletal muscle also may play a role in the pathogenesis of insulin resistance in human obesity (6). Although PTP activity was found to be increased in adipose tissue of obese subjects (7), a previous report suggested a marked impairment of PTPN1 activity in adipose tissue of obese subjects (8). Therefore, the role of PTPN1 in human insulin resistance and obesity is not fully understood. The human PTPN1 gene is encoded on chromosome 20q13.1 (9). In this region, quantitative trait loci have been

1

Nonstandard abbreviations: PTPN1, protein tyrosine phosphatase 1B; LEPR, leptin receptor; TER, trunk-to-extremity skinfold ratio; AIRglucose, acute insulin response to glucose; DI, disposition index; Kg, glucose disappearance index; SI, insulin sensitivity index; FSIGT, frequently sampled intravenous glucose tolerance test.

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Table 1. Genotype and allele frequencies for the PTPN1 polymorphisms Polymorphism IVS6⫹G82A Blacks Whites Pro303Pro Blacks Whites Pro387Leu Blacks Whites

Genotype frequency AA 0.10 (8) 0.29 (55) CC 0.80 (62) 0.86 (163) CC 100.0 (77) 0.98 (187)

AG 0.34 (26) 0.46 (86) CT 0.17 (13) 0.14 (39) CT 0 (0) 0.01 (2)

Allele frequency GG 0.56 (43) 0.25 (48) TT 0.03 (2) 0.01 (3) TT 0.0 (0) 0.0 (1)

A 0.27* 0.52 C 0.89 0.89 C 1.00 0.99

G 0.73 0.48 T 0.11 0.11 T 0.00 0.01

Number of subjects in parentheses. Computations based only on parents. * p ⬍ 0.001 between whites and blacks

found for obesity, fasting serum insulin levels (10), and type 2 diabetes (11). Recent studies have shown associations of some PTPN1 polymorphisms with obesity-related phenotypes (12–14). We studied whether polymorphisms in the PTPN1 gene are associated with body fat distribution and insulin metabolism in the HERITAGE cohort. Due to the possible effect of PTPN1 on leptin signaling (5), we also explored whether there are interactions between PTPN1 and leptin receptor (LEPR) gene markers on insulin metabolism. The genotype distributions and allele frequencies of the three polymorphisms based on parents are presented in Table 1. Blacks exhibited a higher frequency of the G allele of the IVS6⫹G82A polymorphism compared with whites. Genotype frequencies for IVS6⫹G82A and Pro303Pro polymorphisms were in a Hardy-Weinberg equilibrium. In whites, the homozygotes for the G allele of the PTPN1 IVS6⫹G82A polymorphism (G82G subjects) tended to have the highest BMI and had the highest percentage of body fat, plasma leptin, and amount of subcutaneous fat assessed by the sum of eight skinfolds as compared with A82A or A82G subjects (Table 2). However, the trunk-toextremity skinfold ratio (TER), adjusted for age, sex, and fat mass, was lower in G82G than in A82A or A82G subjects. The sum of trunk skinfolds was slightly higher among G82G subjects than in other genotypes, but the difference was not significant after adjustment for total fat mass. The sum of extremity skinfolds was higher in the G82 allele homozygotes than in other genotypes even after adjustment for total fat mass (Table 3). After correction for multiple tests, the significance in the sum of extremity skinfolds before adjustment for total fat mass between the PTPN1 IVS6⫹G82A genotypes re830

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mained. None of the PTPN1 polymorphism was associated with CT-measured abdominal fat phenotypes (data not shown). Glucose metabolism-related variables in whites by PTPN1 polymorphisms are shown in Table 4. Acute insulin response to glucose (AIRglucose) was lower in the A82G subjects than in the A82A or G82G subjects of the G82A polymorphism. In addition, disposition index (DI) was lower in the A82G than in the A82A subjects. The heterozygotes of the Pro387Leu polymorphism showed lower fasting insulin levels and glucose disappearance index (Kg) and slightly lower AIRglucose and DI than the Pro387 allele homozygotes. Plasma glucose did not differ between any of the genotype groups. In blacks, no significant associations were observed. In whites, an interaction was observed between the PTPN1 IVS6⫹G82A and LEPR Gln223Arg gene polymorphisms for insulin sensitivity index (SI) and AIRglucose (Figure 1). The G allele homozygotes for the IVS6⫹G82A and the noncarriers of the Arg allele of the Gln223Arg (GG/Arg⫺) had a lower SI than the subjects with AA⫹AG/ Arg⫺ (p ⫽ 0.038) or GG/Arg⫹ (p ⫽ 0.042). In contrast, AIRglucose was higher in the subjects with GG/Arg⫺ than in those with AA⫹AG/Arg⫺ (p ⫽ 0.025), GG/Arg⫹ (p ⫽ 0.015), or AA⫹AG/Arg⫹ (p ⫽ 0.010). After correction for multiple tests, the interaction effects remained significant. In blacks, no interactions were tested because of lack of statistical power. The rare Pro387Leu variant of the PTPN1 gene was associated with lower plasma fasting insulin levels and lower Kg, an overall index of glucose tolerance. The 387Leu carriers also had a slightly lower DI than the Pro387Pro homozygotes. These findings suggest that 387Leu carriers

PTPN1 Gene and Insulin Metabolism, Ukkola et al.

Table 2. BMI and adiposity phenotypes by PTPN1 IVS6⫹G82A genotypes

BMI (kg/m2) Blacks Whites Percentage body fat Blacks Whites Plasma leptin (ng/mL) Blacks Whites Subcutaneous fat (mm) (sum of eight skinfolds) Blacks Whites TER Blacks Whites Values are means (SE). Adjusted for * p ⫽ 0.009 between AG and GG, p † p ⫽ 0.0078 between AG and GG. ‡ p ⫽ 0.001 between AG and GG, p § p ⫽ 0.022 between AA and GG, p

AA

n

AG

n

GG

n

p (for trend)

26.3 (1.2) 25.8 (0.5)

28 136

28.0 (0.9) 25.6 (0.4)

111 240

28.3 (1.0) 26.7 (0.5)

137 126

NS 0.059

25.9 (2.0) 27.0 (0.9)

24 132

28.8 (1.2) 26.8 (0.9)

91 228

29.4 (1.3) 29.0 (1.1)

115 117

NS 0.031*

10.7 (3.1) 8.4 (1.5)

24 129

11.5 (2.5) 8.0 (1.4)

99 234

11.4 (2.2) 9.9 (2.0)

121 123

NS 0.028†

135.7 (14.4) 150.4 (7.5)

22 123

144.9 (8.9) 145.1 (6.8)

91 225

141.0 (8.3) 165.1 (7.7)

103 116

NS 0.003‡

87 107

NS 0.042§

1.46 (0.13) 1.33 (0.04)

18 120

1.45 (0.06) 1.31 (0.04)

76 213

1.39 (0.06) 1.22 (0.04)

age and sex. The TER was also adjusted for fat mass. ⫽ 0.047 between AA and GG. ⫽ 0.034 between AA and GG ⫽ 0.035 between AG and GG

may have a defect in insulin secretion. The results are in accordance with those described recently in the Danish white population (12), where the rare Pro387Leu variant was associated with a 3.7-fold increased risk of type 2 diabetes. A variant in intron 6 of the PTPN1 gene was also associated with measures of insulin and glucose metabolism. The IVS6 A82G subjects showed the lowest AIRglucose, an index of insulin secretion, as well as the lowest DI. These findings support the notion that A82G subjects may have, at least to some extent, ␤-cell dysfunction. The glucose metabolism-related indices were adjusted for total fat. Therefore, the associations with insulin metabolism were not dependent on the amount of total fat. Evidence for an involvement of PTPN1 on the development of animal obesity was provided by the recent observation that a decreased PTPN1 protein level in fat tissue leads to lower levels of adiposity (15). Studies on PTPN1 in human obesity have, however, given contradictory results. In the present study, the IVS6⫹G82A variant was associated with accumulation of subcutaneous fat on the limbs. It can be speculated that the polymorphism may be associated with a variation in PTPN1 activity. PTP activity has been shown to be increased in adipose tissue of obese subjects

(7). The activity of soluble PTP in skeletal muscle has also been shown to be higher in nondiabetic obese (6) and insulin-resistant (16) subjects. What are the functional consequences of the studied variants? The Pro387Leu variant has been shown to be related to impaired in vitro serine phosphorylation of the PTPN1 peptide (12). The IVS6⫹G82A polymorphism is located near exon 6, the active site of the gene. It is possible that there is a functional polymorphism/mutation in linkage disequilibrium with the IVS6⫹G82A polymorphism in the PTPN1 gene or elsewhere that is responsible for the observed association. It has been recently documented that PTPN1 negatively regulates leptin signaling (5), which, in part, may explain the low adiposity of the PTPN1 knockout mice who have increased leptin sensitivity. We observed interactions between LEPR and PTPN1 genes. Interestingly, LEPR Gln223Arg carrier status influenced the association between the PTPN1 IVS6⫹G82A polymorphism and insulin phenotypes. This interaction was characterized by decreased insulin sensitivity in the subjects who were homozygous for the G allele of the PTPN1 IVS2⫹G82A polymorphism and noncarriers of Arg allele of the LEPR Gln223Arg polymorphism. In addition, these subjects had an increased AIRglucose, possible due to OBESITY RESEARCH Vol. 13 No. 5 May 2005

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Table 3. Subcutaneous fat distribution by PTPN1 IVS6⫹G82A genotypes

Sum of trunk skinfolds (mm) Blacks Whites Sum of extremity skinfolds (mm) Blacks Whites

AA

n

AG

n

GG

n

p (for trend)

79.0 (8.4) 82.3 (4.5) 80.3 (2.5)†

25 124 121

81.7 (5.7) 79.1 (3.9) 78.0 (2.2)†

94 228 216

81.2 (5.9) 87.8 (4.3) 79.7 (2.7)†

114 118 109

NS 0.031* NS

59.1 (5.7) 68.8 (3.3) 68.4 (1.8)†

23 130 127

67.3 (4.1) 67.2 (3.1) 66.6 (1.7)†

101 236 224

68.3 (3.7) 77.4 (3.7) 71.6 (2.0)†

120 120 111

NS 0.002‡ 0.026§

Values are means (SE). Adjusted for age and sex. * p ⫽ 0.009 between AG and GG. † In whites, values adjusted for total fat mass. ‡ p ⫽ 0.0004 between AG and GG, p ⫽ 0.011 between AA and GG. § p ⫽ 0.008 between AG and GG.

compensation of insulin resistance. Therefore, the DI was not different between the genotype combinations. Thus, the genotype combination did not eventually lead to deteriorated overall insulin metabolism indicators in those subjects whose ␤-cell function was intact.

In conclusion, variation at PTPN1 IVS6⫹G82A was associated with body composition with G82G homozygotes showing higher levels of all measures of adiposity. G82 allele heterozygotes are potentially at higher risk for type 2 diabetes. Gene-gene interactions between the PTPN1 and

Table 4. Glucose metabolism-related indices by PTPN1 polymorphisms in whites Polymorphisms IVS6⫹G82A AA (n ⫽ 123 to 127) AG (n ⫽ 213 to 224) GG (n ⫽ 111 to 116) Pro303Pro CT (n ⫽ 377 to 393) CC (n ⫽ 70 to 73) Pro387Leu Pro387Leu (n ⫽ 11) Pro387Pro (n ⫽ 440 to 457)

Fasting insulin (pM)

Kg (%/min)

SI (ⴛ10ⴚ5/min/ per picomolar)

AIRglucose (pM/10 min)

DI

54.9 (7.3) 52.7 (7.2) 53.4 (8.4) NS

1.59 (0.06) 1.49 (0.06) 1.56 (0.07) NS

3.3 (0.5) 3.4 (0.5) 3.5 (0.4) NS

61.5 (12.9) 47.6 (9.0) 56.0 (12.1) 0.005*

201.9 (34.3) 169.0 (34.5) 196.8 (45.0) 0.040†

53.9 (6.6) 49.8 (12.5) NS

1.54 (0.05) 1.52 (0.09) NS

3.4 (0.4) 3.5 (0.7) NS

53.7 (8.4) 50.9 (20.9) NS

184.8 (30.7) 187.5 (58.8) NS

43.2 (12.9) 53.5 (19.6) 0.035

1.15 (0.08) 1.54 (0.05) 0.038

3.3 (2.1) 3.4 (0.8) NS

47.7 (25.1) 53.5 (18.0) NS

166.8 (77.9) 185.6 (64.9) NS

Values are means (logarithmic back-transformed except for Kg) (SE) adjusted for age, sex, and fat mass. * p ⫽ 0.002 between AA and AG, p ⫽ 0.035 between AG and GG. † p ⫽ 0.019 between AA and AG.

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Research Methods and Procedures Subjects The HERITAGE Family Study is a multicenter clinical trial conducted at five institutions (17). The sample for the present study consists of 778 subjects, including 502 whites and 276 blacks. To be enrolled in the study, the individuals were required to be in good health (i.e., free of diabetes, cardiovascular diseases, or other chronic diseases) and to be sedentary at baseline (defined as no regular strenuous physical activity over the previous 6 months). The study protocol had been previously approved by each of the Institutional Review Boards of the HERITAGE Family Study research consortium. Written informed consent was obtained from each participant.

Figure 1: Glucose metabolism-related indices in relation to PTPN1 IVS2⫹G82A and LEPR Gln223Arg genotypes in whites. All of the indices are adjusted for total fat mass, age, and sex. (*) Significant between GG/Arg⫺ and AA⫹AG/Arg⫺ (p ⫽ 0.038) or GG/Arg⫹ (p ⫽ 0.042). (**) Significant between GG/Arg⫺ vs. AA⫹AG/Arg⫹ (p ⫽ 0.025), GG/Arg⫹ (p ⫽ 0.015), or AA⫹AG/Arg⫺ (p ⫽ 0.0096).

LEPR genes contributed to the phenotypic variability in insulin sensitivity. The PTPN1 Pro387Leu variant may be associated with lower glucose tolerance.

Phenotype Measurements In the present study, analyses were performed only with baseline phenotype data. Body composition measures included hydrostatic weight, residual volume, and body density, from which fat mass and fat-free mass were estimated (18). The sum of eight skinfolds was used to assess the level of subcutaneous fat. The trunk-to-extremity skinfolds ratio (⫽[subscapular ⫹ suprailiac ⫹ abdominal ⫹ midaxillary]/ [biceps ⫹ triceps ⫹ medial calf ⫹ thigh]) was used as an indicator of the propensity to store fat in the truncal-abdominal area relative to the limbs. Abdominal fat was quantified by computerized tomography. Plasma leptin level (in nanograms per milliliter) was measured by radioimmunoassay (Linco, St. Charles, MO). A frequently sampled intravenous glucose tolerance test (FSIGT) was performed in the morning after an overnight (12-hour) fast as described earlier (19). The Kg, used as a measure of overall glucose tolerance, was defined as an estimate of the disappearance rate (percentage per minute) of plasma glucose based on the slope of the line derived from least-squares regression of the natural logarithm of plasma glucose on time from 10 to 60 minutes during the FSIGT. The AIRGlucose was computed as the incremental integrated area under the insulin curve for the first 10 minutes of the FSIGT and was used as an index of insulin secretion. The SI represents the increase in net fractional glucose clearance rate per unit change in plasma insulin concentration after the intravenous glucose load. The DI was derived as the product of SI and AIRGlucose and is a measure of the activity of the ␤ cells adjusted for insulin resistance. All these variables were derived from the Minimal Model analysis. Because of skewed distributions, AIRGlucose, SI, and DI were transformed using a natural logarithm. Genotype Determinations Genomic DNA was isolated from lymphoblastoid cell lines by the proteinase K and phenol/chloroform technique. The IVS6⫹G82A and Pro 303Pro polymorphisms in the OBESITY RESEARCH Vol. 13 No. 5 May 2005

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PTPN1 gene were typed as we have reported earlier (14). The genotyping of Pro387Leu polymorphism in tyrosine phosphatase 1B gene was performed as described earlier (12). The polymerase chain reaction analysis of the LEPR Gln223Arg polymorphism was based on an earlier report (20). Statistical Analysis All analyses were performed with the SAS Statistical Software Package (SAS Institute Inc., Cary, NC). A ␹2 test was performed to determine whether the genotype frequencies were in the Hardy-Weinberg equilibrium. Association analyses were carried out using a MIXED procedure in the SAS software package. Nonindependence among family members was adjusted for using a sandwich estimator, which asymptotically yields the same parameter estimates as ordinary least squares or regression methods, but the SEs and, consequently, hypothesis tests are adjusted for the within-family dependencies. The method is general, assuming the same degree of dependency among all members within a family. Gene-gene interactions were tested with the MIXED procedure by including the main effects of the gene, interaction terms, and covariates in the same model. p values were corrected for the number of multiple comparisons through the Bonferroni method.

Acknowledgments The HERITAGE Family Study is supported by National Heart, Lung, and Blood Institute Grants HL-45670 (to C.B.), HL-47323 (to A.S.L.), HL-47317 (to D.C.R.), HL47327 (to J.S.S.), and HL-47321 (to J.H.W.). A.S.L. is partially supported by the Henry L. Taylor endowed Professorship in Exercise Science and Health Enhancement. C.B. is supported, in part, by the George A. Bray Chair in Nutrition. This work was supported, in part, by a grant from the Medical Council of the Academy of Finland and the Finnish Foundation for Cardiovascular Research. We thank Heidi Ha¨ikio¨ for her contributions to this study. References 1. Neel BG, Tonks NK. Protein tyrosine phosphatases in signal transduction. Curr Opin Cell Biol. 1997;9:193–204. 2. Byon JCH, Kusari AB, Kusari J. Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction. Mol Cell Biochem. 1998;182:101– 8. 3. Ahmad F, Li PM, Meyerovitch J, Goldstein BJ. Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway. J Biol Chem. 1995;270:20503– 8. 4. Elchebly M, Payette P, Michaliszyn E, et al. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science. 1999;283: 1544 – 8. 5. Kaszubska W, Falls HD, Schaefer VG, et al. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol Cell Endocrinol. 2002;195:109 – 18.

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